Small molecules used to increase cell death

ABSTRACT

The invention features methods for increasing cell death. The invention also features compounds used to increase cell death. The invention further features methods for identifying compounds that increase cell death.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/802,902, filed Mar. 16, 2004, which is a divisional of U.S.application Ser. No. 10/196,080, filed Jul. 16, 2002, which is adivisional of U.S. application Ser. No. 09/736,502, filed Dec. 13, 2000,which claims priority from U.S. Provisional Application Ser. No.60/170,329, filed Dec. 13, 1999, each of which is hereby incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported in part by the Government under Grant No. DAMD17-98-1-8102 from the Department of the Army. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

In general, the invention features methods and compounds for increasingcell death.

Cell growth is a tightly regulated process. When the body has no needfor new cells, but cells nonetheless divide in an unregulated manner,the result is cancer. Cancer therapies are directed at controlling therapid proliferation of cells and/or controlling the differentiation rateof cells, as an undifferentiated cell is highly proliferative. One wayin which the proliferation of cancer cells may be controlled is bykilling such unregulated dividing cells.

The family of Bcl-2 proteins plays a central role in the regulation ofcell life and death, acting by modulating apoptosis, a specific type ofcell death. Some members of this family, for example, Bax, Bad, and Bakpromote apoptosis, while other members of the family, for example,Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 inhibit apoptosis. The precise mechanismby which the various Bcl-2 family members promote either cell viabilityor cell death has not yet been resolved.

One method for treating cancer involves controlling the expressionand/or activity of Bcl-2 family member proteins. In particular, methodsthat decrease the expression or activity of anti-apoptotic Bcl-2 familymembers or increase the expression or activity of pro-apoptotic Bcl-2family members would be useful for treating cancer.

SUMMARY OF THE INVENTION

The present invention features methods and compounds for disrupting aninteraction between a polypeptide containing a Bcl-2-homology-3 domainand another polypeptide, and for increasing cell death. The compounds ofthe present invention may be used as therapeutics to increase cell deathin a desired cell, such as a cancer cell. These compounds arecharacterized by their ability to inhibit heterodimerization betweenpro-apoptotic and anti-apoptotic members of the Bcl-2 family ofproteins. Identified compounds may be especially useful in treatingcancers that overexpress Bcl-2 protein family members.

Accordingly, in a first aspect, the invention features a chemicalcompound in a pharmaceutically acceptable carrier having the formula:

where each of R₁, R₂, R₄, and R₅ is independently selected from thegroup consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R₃ isselected from the group consisting of N(CH₃)₂, phenyl, hydroxyl,alkoxyl, and halogens; R₆ is selected from the group consisting ofCH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₃; R₇ is either hydrogen oran alkyl group; and the bond (a) is either a single or double bond.

In a preferred embodiment of the above aspect of the invention, theheterocyclic ring of the compound is substituted with a benzyl ring.

In another preferred embodiment, in the compound, each of R₁, R₂, R₄,and R₅ are hydrogen; R₃ is bromine; R₆ is CH(CH₃)₂; R₇ is hydrogen; andthe bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is chlorine; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₃, R₄, and R₅ are hydrogen; R₆ is CH(CH₃)₂; R₇ is hydrogen; and thebond (a) is a double bond.

In still another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is N(CH₃)₂; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In other embodiments, the alkoxyl group of R₁, R₂, R₄, R₅, or R₃contains 10 or fewer carbons. Preferably the alkoxyl group of R₁, R₂,R₄, R₅, or R₃ contains 4 or fewer carbons. Most preferably the alkoxylgroup of R₁, R₂, R₄, R₅, or R₃ is a methoxyl group.

In yet other embodiments of the above aspect of the invention, if R₁ ishydrogen, then R₂, R₄, or R₅ is not hydrogen; or R₃ is not bromine orchlorine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a)is not a double bond. If R₂ is hydrogen, then R₁, R₄, or R₅ is nothydrogen; or R₃ is not bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇is not hydrogen; or the bond (a) is not a double bond. If R₄ ishydrogen, then R₁, R₂, or R₅ is not hydrogen; or R₃ is not bromine orchlorine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a)is not a double bond. If R₅ is hydrogen, then R₁, R₂, or R₄ is nothydrogen; or R₃ is not bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇is not hydrogen; or the bond (a) is not a double bond. If R₃ is bromineor chlorine, then R₁, R₂, R₄, or R₅ is not hydrogen; or R₆ is notCH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not a double bond.If R₆ is CH(CH₃)₂, then R₁, R₂, R₄, or R₅ is not hydrogen; or R₃ is notchlorine or bromine; or R₇ is not hydrogen; or the bond (a) is not adouble bond. If R₇ is hydrogen, then R₁, R₂, R₄, or R₅ is not hydrogen;or R₃ is not chlorine or bromine; or R₆ is not CH(CH₃)₂; or the bond (a)is not a double bond. If the bond (a) is a double bond, then R₁, R₂, R₄,or R₅ is not hydrogen; or R₃ is not chlorine or bromine; or R₆ is notCH(CH₃)₂; or R₇ is not hydrogen.

In a second aspect, the invention features a chemical compound in apharmaceutically acceptable carrier having the formula:

where each of R₁, R₂, R₄, and R₅ is, independently, hydrogen, a halogen,or a phenyl group; and R₃ is hydrogen or an alkyl group.

In a preferred embodiment of the second aspect of the invention, in thecompound, each of R₁, R₄, and R₅ is chlorine; R₂ is bromine; and R₃ ishydrogen.

In another preferred embodiment of the second aspect of the invention,in the compound, each of R₁, R₄, and R₅ is chlorine; R₂ is iodine; andR₃ is hydrogen.

In yet another preferred embodiment of the second aspect of theinvention, in the compound, R₁ and R₂ are iodine, R₄, and R₅ arechlorine; and R₃ is hydrogen.

In other embodiments of the second aspect of the invention, if R₁ ischlorine, then R₄ or R₅ is not chlorine; or R₂ is not bromine; or R₃ isnot hydrogen. If R₄ is chlorine, then R₁ or R₅ is not chlorine; or R₂ isnot bromine; or R₃ is not hydrogen. If R₅ is chlorine, then R₁ or R₄ isnot chlorine; or R₂ is not bromine; or R₃is not hydrogen. If R₂ isbromine, then R₁, R₄, or R₅ is not chlorine; or R₃ is not hydrogen. IfR₃ is hydrogen, then R₁, R₄, or R₅ is not chlorine; or R₂ is notbromine.

In a third aspect, the invention features a method for increasing celldeath, involving the steps of:

(a) providing a cell predicted to be resistant to cell death, or to beat risk for resisting cell death; and

(b) contacting the cell with a chemical compound having the formula:

where each of R₁, R₂, R₄, and R₅ is independently selected from thegroup consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R₃ isselected from the group consisting of N(CH₃)₂, phenyl, hydroxyl,methoxyl, and halogens; R₆ is selected from the group consisting ofCH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₃; R₇ is either hydrogen oran alkyl group; and the bond (a) is either a single or double bond.

In a preferred embodiment of the above aspect of the invention, in thecompound, the heterocyclic ring of the compound is substituted with abenzyl ring.

In another preferred embodiment, in the compound, each of R₁, R₂, R₄,and R₅ are hydrogen; R₃ is bromine; R₆ is CH(CH₃)₂; R₇ is hydrogen; andthe bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is chlorine; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₃, R₄, and R₅ are hydrogen; R₆ is CH(CH₃)₂; R₇ is hydrogen; and thebond (a) is a double bond.

In still another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is N(CH₃)₂; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In other embodiments, in the compound, the alkoxyl group of R₁, R₂, R₄,R₅, or R₃ contains 10 or fewer carbons. Preferably the alkoxyl group ofR₁, R₂, R₄, R₅, or R₃ contains 4 or fewer carbons. Most preferably thealkoxyl group of R₁, R₂, R₄, R₅, or R₃ is a methoxyl group.

In yet other embodiments of the above aspect of the invention, in thecompound, if R₁ is hydrogen, then R₂, R₄, or R₅ is not hydrogen; or R₃is not bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇ is nothydrogen; or the bond (a) is not a double bond. If R₂ is hydrogen, thenR₁, R₄, or R₅ is not hydrogen; or R₃ is not bromine or chlorine; or R₆is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not a doublebond. If R₄ is hydrogen, then R₁, R₂, or R₅ is not hydrogen; or R₃ isnot bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen;or the bond (a) is not a double bond. If R₅ is hydrogen, then R₁, R₂, orR₄ is not hydrogen; or R₃ is not bromine or chlorine; or R₆ is notCH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not a double bond.If R₃ is bromine or chlorine, then R₁, R₂, R₄, or R₅ is not hydrogen; orR₆ is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not adouble bond. If R₆ is CH(CH₃)₂, then R₁, R₂, R₄, or R₅ is not hydrogen;or R₃ is not chlorine or bromine; or R₇ is not hydrogen; or the bond (a)is not a double bond. If R₇ is hydrogen, then R₁, R₂, R₄, or R₅ is nothydrogen; or R₃ is not chlorine or bromine; or R₆ is not CH(CH₃)₂; orthe bond (a) is not a double bond. If the bond (a) is a double bond,then R₁, R₂, R₄, or R₅ is not hydrogen; or R₃ is not chlorine orbromine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen.

In a fourth aspect, the invention features a method for increasing celldeath, said method involving the steps of:

(a) providing a cell predicted to be resistant to cell death, or to beat risk for resisting cell death; and

(b) contacting the cell with a chemical compound having the formula:

where each of R₁, R₂, R₄, and R₅ is, independently, hydrogen, a halogen,or a phenyl group; and R₃ is hydrogen or an alkyl group.

In a preferred embodiment of the fourth aspect of the invention, in thecompound, each of R₁, R₄, and R₅ is chlorine; R₂ is bromine; and R₃ ishydrogen.

In another preferred embodiment of the fourth aspect of the invention,in the compound, each of R₁, R₄, and R₅ is chlorine; R₂ is iodine; andR₃ is hydrogen.

In yet another preferred embodiment of the fourth aspect of theinvention, in the compound, R₁ and R₂ are iodine, R₄, and R₅ arechlorine; and R₃ is hydrogen.

In other embodiments of the fourth aspect of the invention, in thecompound, if R₁ is chlorine, then R₄ or R₅ is not chlorine; or R₂ is notbromine; or R₃ is not hydrogen. If R₄ is chlorine, then R₁ or R₅ is notchlorine; or R₂ is not bromine; or R₃ is not hydrogen. If R₅ ischlorine, then R₁ or R₄is not chlorine; or R₂ is not bromine; or R₃ isnot hydrogen. If R₂ is bromine, then R₁, R₄, or R₅ is not chlorine; orR₃ is not hydrogen. If R₃ is hydrogen, then R₁, R₄, or R₅ is notchlorine; or R₂ is not bromine.

In one embodiment of the third or fourth aspect of the invention, thecell expresses a pro-apoptotic and/or anti-apoptotic protein. Preferablythe pro-apoptotic protein is selected from the group consisting ofpro-apoptotic proteins containing a Bcl-2-homology-domain-3, such asBax, Bak, Bok, Bad, Bid, Bik, Bim, or Hrk. In another embodiment, theanti-apoptotic protein is chosen from the group consisting of Bcl-2,Bcl-xL, Mcl-1, and Bcl-w. In another embodiment, the compound issubstantially pure. In another embodiment, the compound is in apharmaceutically acceptable carrier.

In another embodiment of the third and fourth aspects of the invention,the cell is mammalian. Preferably the cell is a rodent cell, such as amouse or rat cell. Most preferably, the cell is a human cell. In anotherembodiment, the cell is a cancer cell.

In a fifth aspect, the invention features a method for treating acondition in a subject, involving administering a chemical compoundhaving the formula:

where each of R₁, R₂, R₄, and R₅ is independently selected from thegroup consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R₃ isselected from the group consisting of N(CH₃)₂, phenyl, alkoxyl,hydroxyl, and halogens; R₃ is selected from the group consisting ofCH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₃; R₇ is either hydrogen oran alkyl group; and the bond (a) is either a single or double bond.

In a preferred embodiment of the above aspect of the invention, in thecompound, the heterocyclic ring of the compound is substituted with abenzyl ring.

In another preferred embodiment, in the compound, each of R₁, R₂, R₄,and R₅ are hydrogen; R₃ is bromine; R₆ is CH(CH₃)₂; R₇ is hydrogen; andthe bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is chlorine; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In yet another preferred embodiment, in the compound, each of R₁, R₂,R₃,R₄, and R₅ are hydrogen; R₆ is CH(CH₃)₂; R₇ is hydrogen; and the bond(a) is a double bond.

In still another preferred embodiment, in the compound, each of R₁, R₂,R₄, and R₅ are hydrogen; R₃ is N(CH₃)₂; R₆ is CH(CH₃)₂; R₇ is hydrogen;and the bond (a) is a double bond.

In other embodiments, in the compound, the alkoxyl group of R₁, R₂, R₄,R₅, or R₃ contains 10 or fewer carbons. Preferably the alkoxyl group ofR₁, R₂, R₄, R₅, or R₃ contains 4 or fewer carbons. Most preferably thealkoxyl group of R₁, R₂, R₄, R₅, or R₃ is a methoxyl group.

In yet other embodiments of the above aspect of the invention, in thecompound, if R₁ is hydrogen, then R₂, R₄, or R₅ is not hydrogen; or R₃is not bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇ is nothydrogen; or the bond (a) is not a double bond. If R₂ is hydrogen, thenR₁, R₄, or R₅ is not hydrogen; or R₃ is not bromine or chlorine; or R₆is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not a doublebond. If R₄ is hydrogen, then R₁, R₂, or R₅ is not hydrogen; or R₃ isnot bromine or chlorine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen;or the bond (a) is not a double bond. If R₅ is hydrogen, then R₁, R₂, orR₄ is not hydrogen; or R₃ is not bromine or chlorine; or R₆ is notCH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not a double bond.If R₃ is bromine or chlorine, then R₁, R₂, R₄, or R₅ is not hydrogen; orR₆ is not CH(CH₃)₂; or R₇ is not hydrogen; or the bond (a) is not adouble bond. If R₆ is CH(CH₃)₂, then R₁, R₂, R₄, or R₅ is not hydrogen;or R₃ is not chlorine or bromine; or R₇ is not hydrogen; or the bond (a)is not a double bond. If R₇ is hydrogen, then R₁, R₂, R₄, or R₅ is nothydrogen; or R₃ is not chlorine or bromine; or R₆ is not CH(CH₃)₂; orthe bond (a) is not a double bond. If the bond (a) is a double bond,then R₁, R₂, R₄, or R₅ is not hydrogen; or R₃ is not chlorine orbromine; or R₆ is not CH(CH₃)₂; or R₇ is not hydrogen.

In a sixth aspect, the invention features a method for treating acondition in a subject, involving administering a chemical compoundhaving the formula:

where each of R₁, R₂, R₄, and R₅ is, independently, hydrogen, a halogen,or a phenyl group; and R₃ is hydrogen or an alkyl group.

In a preferred embodiment of the sixth aspect of the invention, in thecompound, each of R₁, R₄, and R₅ is chlorine; R₂ is bromine; and R₃ ishydrogen.

In another preferred embodiment of the sixth aspect of the invention, inthe compound, each of R₁, R₄, and R₅ is chlorine; R₂ is iodine; and R₃is hydrogen.

In yet another preferred embodiment of the sixth aspect of theinvention, in the compound, R₁ and R₂ are iodine, R₄, and R₅ arechlorine; and R₃ is hydrogen.

In other embodiments of the sixth aspect of the invention, in thecompound, if R₁ is chlorine, then R₄ or R₅ is not chlorine; or R₂ is notbromine; or R₃ is not hydrogen. If R₄ is chlorine, then R₁ or R₅ is notchlorine; or R₂ is not bromine; or R₃ is not hydrogen. If R₅ ischlorine, then R₁ or R₄ is not chlorine; or R₂ is not bromine; or R₃ isnot hydrogen. If R₂ is bromine, then R₁, R₄, or R₅ is not chlorine; orR₃ is not hydrogen. If R₃ is hydrogen, then R₁, R₄, or R₅ is notchlorine; or R₂ is not bromine.

In another embodiment of the fifth or sixth aspect of the invention, thecondition is any condition in which the occurrence of cell death is toolow. Preferably the condition is cancer, such as prostate cancer, breastcancer, gastrointestinal cancer, non-small cell lung cancer, coloncancer, melanoma, ovarian cancer, stomach cancer, or a brain tumor, or aleukemia, lymphoma, or carcinoma.

In another embodiment, the subject is a mammal. Preferably the subjectis a rodent, such as a mouse or rat. Most preferably the subject is ahuman.

In another embodiment of the fifth or sixth aspect of the invention, atleast two of the compounds are administered, preferably, simultaneously.

In a seventh aspect, the invention features a method for identifying acompound that disrupts an interaction between a first polypeptidecontaining a Bcl-2-homology-3 domain and a second polypeptide, involvingthe steps of: providing a test compound, a first polypeptide containinga Bcl-2-homology-3 domain, and a second polypeptide; combining the testcompound, first polypeptide, and second polypeptide; and measuring theinteraction between the first and second polypeptides, relative to acontrol that comprises only the first and second polypeptides, where themeasuring is done using a fluorescence polarization assay, and where adecrease in the interaction between the first and second polypeptidesidentifies the test compound as disrupting an interaction between thefirst and second polypeptides.

In an eighth aspect, the invention features a method for identifying acompound that increases cell death, involving the steps of: contacting acell with a test compound that disrupts the interaction between a firstpolypeptide containing a Bcl-2-homology-3 domain and a secondpolypeptide; and measuring cell death relative to a control cell, wherethe measuring is done using a fluorescence polarization assay, and wherean increase in cell death indicates that the test compound increasescell death.

In one embodiment of the seventh or eighth aspect of the invention, thefirst polypeptide is chosen from the group consisting of Bax, Bak, Bok,Bad, Bid, Bik, Bim, and Hrk.

By “increasing cell death” is meant increasing the number of cells thatundergo cell death relative to a control cell that is not contacted withany test compounds. Preferably cell death is increased 10% relative to acontrol. More preferably cell death is increased 50% relative to acontrol. Most preferably cell death is increased is increased 90%relative to a control.

Cell death may be increased by contacting a cell with a test compound.An increase in cell death may be identified by determining the ATP levelin a cell that has been contacted with a test compound, such as a smallmolecule from a chemical library, and comparing it to the ATP level in acontrol cell, for example, according to the methods of Crouch et al. (J.Immunol. Methods 160:81-8, 1993) Storer et al. (Mutat. Res. 368:59-101,1996) or Cree et al. (Toxicol. In Vitro 11:553-556, 1997). Cell death isincreased when the ATP level of a cell contacted with a test compounddecreases more than the ATP level of a control cell. Cell death may alsobe measured using any of the assays described herein.

By “the occurrence of cell death is too low” or “resistant to celldeath” is meant that a cell or a population of cells does not undergocell death under appropriate conditions. For example, normally a cellwill die upon exposure to cytotoxic agents, such as chemotherapeuticagents or ionizing radiation. However, when the occurrence of cell deathis too low, for example, in a subject having cancer, the cell or apopulation of cells may not undergo cell death in response to contactwith cytotoxic agents. In addition, the occurrence of cell death may betoo low when the number of proliferating cells exceeds the number ofcells undergoing cell death, as occurs in cancer when such cells do notproperly differentiate.

By “cell death” is meant the death of a cell by either apoptosis ornecrosis. Cell death may be characterized by cellular ATP depletion.Preferably the cell is depleted of ATP 10% relative to a control cell.More preferably the cell is depleted of ATP 50% relative to a controlcell. Most preferably the cell is depleted of ATP 90% relative to acontrol cell. The level of cell death may be measured by determining theamount of ATP in a cell.

By “test compound” is meant a chemical, be it naturally-occurring orartificially-derived, that is surveyed for its ability to modulate thelevel of cell death by employing one of the assay methods describedherein. Test compounds may include, for example, peptides, polypeptides,synthesized organic molecules, naturally-occurring organic molecules,nucleic acid molecules, and components thereof. Test compounds alsoinclude salts of any of the above chemicals.

By “apoptosis” is meant cell death characterized by any of the followingproperties: nuclear condensation, DNA fragmentation, membrane blebbing,or cell shrinkage.

By an “anti-apoptotic-protein” is meant a protein which when expressedin a cell decreases cell death, as compared to a cell that does notexpress the anti-apoptotic protein. Preferably cell death in the cellcontaining the anti-apoptotic protein is decreased 10% relative to acontrol. More preferably cell death in the cell containing theanti-apoptotic protein is decreased 50% relative to a control. Mostpreferably cell death in the cell containing the anti-apoptotic proteinis decreased 90% relative to a control.

By a “pro-apoptotic protein” is meant a protein that when expressed in acell increases cell death, as compared to a cell that does not expressthe pro-apoptotic protein.

Preferably cell death in the cell containing the pro-apoptotic proteinis increased 10% relative to a control. More preferably cell death inthe cell containing the pro-apoptotic protein is increased 50% relativeto a control. Most preferably cell death in the cell containing thepro-apoptotic protein is increased 90% relative to a control.

By “interacts” is meant a compound that recognizes and binds to ananti-apoptotic protein but which does not substantially recognize andbind to other molecules in a sample.

By “disrupts an interaction” is meant that a test compound decreases theability of two polypeptides to interact with each other. Preferably thedisruption results in a 50% decrease in the ability of the polypeptidesto interact with each other. More preferably disruption results in a 75%decrease in the ability of the polypeptides to interact with each other.Most preferably the disruption results in a 99% decrease in the abilityof the polypeptides to interact with each other.

As used herein, by “substantially pure” is meant a compound that is atleast 60%, by weight, free from proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably thepreparation is at least 75%, more preferably 90%, and most preferably atleast 99%, by weight, said compound, e.g., a compound from a chemicallibrary. A purified compound may be obtained using methods known tothose in the fields of medicinal and organic chemistry.

By “containing a Bcl-2-homology-3 domain” or “containing a BH3 domain”or a “BH3 peptide” is meant a polypeptide that is substantiallyidentical to the amino acid sequence LRRIGDEF (SEQ ID NO: 1).

By “substantially identical” is meant a polypeptide exhibiting at least60%, preferably 85%, more preferably 90%, and most preferably 95%homology to a reference amino acid sequence.

By “treating” is meant to submit or subject an animal, cell, lysate orextract derived from a cell, or a molecule derived from a cell to acompound that increases cell death.

By “condition” is meant a state of being or feeling. Conditions include,but are not limited to, cancer, for example, prostate cancer, breastcancer, gastrointestinal cancer, non-small cell lung cancer, coloncancer, melanoma, ovarian cancer, stomach cancer, or a brain tumor, or aleukemia, lymphoma, or carcinoma, or the symptoms associated withcancer.

By a “dosage sufficient to increase cell death” is meant an amount of achemical compound or small molecule which when administered to a subjectwill increase cell death. Preferably cell death is increased in thesubject 10% relative to an untreated subject. More preferably cell deathis increased in the subject 50% relative to an untreated subject. Mostpreferably cell death is increased in the subject 90% relative to anuntreated subject.

By a “derivative” is meant a structural derivative having a chemicalmodification of the compound which does not increase the ultimate levelof cell death, but which does enhance bioavailability, solubility, orstability in vivo or ex vivo or which reduces the toxicity or dosagerequired. Such modifications are known to those skilled in the field ofmedicinal chemistry.

As used herein, by “measuring cell death” is meant determining if a cellis dying in the presence of a compound compared to a cell that is not inthe presence of the compound (control cell). Cell death can be measuredby determining cellular ATP levels, wherein a cell that is undergoingcell death has a decreased level of cellular ATP compared to a controlcell. Cell death may also be measured by staining with a vital dye, forexample, trypan blue, wherein a cell that is dead will be stained withthe vital dye, and a cell that is not dead will not be stained with thedye. Cell death can also be measured by contacting a cell with Hoeschtstain and viewing it for morphological indications of cell death. Suchindications include nuclear fragmentation. Other assays for measuringcell death are described herein.

By “fluorescence polarization assay” is meant an assay in which aninteraction between two polypeptides is measured. In this assay, onepolypeptide is labeled with a fluorescent tag, and this polypeptideemits nonpolarized light when excited with polarized light. Upon aninteraction of the tagged polypeptide with another polypeptide, thepolarization of emitted light is increased, and this increasedpolarization of light can be detected.

The present invention provides a number of advantages. For example, themethods described herein allow for an increase in cell death or adisruption of the interaction between Bcl-2 family members. Theinvention also provides compounds and methods for treating diseases inwhich a cell is resistant to cell death. These compounds and methods canbe used to treat conditions such as cancer.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the chemical structure ofchemical compound ID number 275805 (BH3I-1) from the CheiBridge chemicalcompound library.

FIG. 2 is a schematic representation of the chemical structure ofchemical compound ID number 282986 (BH3-I-1′) from the Chemnridgechemical compound library.

FIG. 3 is a schematic representation of the chemical structure ofchemical compound ID number 175362 (BH3I-2) from the ChemBridge chemicalcompound library.

FIG. 4 shows two schematic representations of the structures andchemical names of the compounds selected in the screen. The Ki valuesfor the inhibition of Bak/Bcl-xL-His₆ interaction, determined usingfluorescence polarization and NMR titration assays, are shown with thestandard deviation values.

FIG. 5A is a graph showing the binding of increasing amounts of the BakBH3 peptide to SELDI surfaces modified with 2 pmoles of Bcl-xL-His₆ inthe presence or absence of the indicated amounts of BH3Is. The insert ofthis figure shows examples of actual mass spectrometry data.

FIG. 5B is a graph showing the effects of BH3Is on the binding of BakBH3 peptide to Bcl-2. Bak BH3 peptide was incubated with SELDI surfacesmodified with 5 pmoles of GST-Bcl-2 in the presence or absence of theindicated concentrations of BH3Is. As a negative control (---), Bak BH3peptide was incubated with SELDI surface without Bcl-xL/Bcl-2 proteins.

FIG. 5C is an image of an autoradiograph of tBid expression andcoomassie blue images of Bcl-xL from pulldown experiments.Bcl-xL-His₆-containing agarose beads were pre-incubated with theindicated concentrations of BH3I-2, and then with ³⁵S labeled in vitrotranslated tbid. Ni-NTA (Qiagen) beads without protein were used as anegative control (blank).

FIG. 5D is an image of an autoradiograph of tBid expression andcoomassie blue images of Bcl-xL from pulldown experiments.Bcl-xL-His₆-containing agarose beads were pre-incubated with theindicated concentrations of BH3I-2, BH3I-1, and Bak BH3 peptide, andthen with ³⁵S labeled in vitro translated tBid. Ni-NTA (Qiagen) beadswithout protein were used as a negative control (blank). FIG. 5E is animage of an autoradiograph of U2AF⁶⁵ expression from pulldownexperiments. U2AF³⁵-His₆-containing agarose beads were pre-incubatedwith the indicated concentrations of BH3I-2, BH3I-1, and then with ³⁵Slabeled in vitro translated U2AF⁶⁵. Ni-NTA (Qiagen) beads withoutprotein were used as a negative control (blank).

FIG. 6A is a series of images showing the viability of cells treatedwith various BH3Is.

FIG. 6B is a set of graphs showing the percentage of subG 1 cells in apopulation of cells left untreated (left), treated with BH3I-1 (center),or treated with BH3I-2 (right).

FIG. 6C is a series of FACS analysis graphs showing Annexin V stainingof Jurkat cells. Jurkat cells were treated with BH3I-1 (100 μM) orBH3I-2 (30 μM) and were stained 4 or 6 hr later with Annexin V-EGFP/PI.The relative amounts of live, apoptotic, and late apoptotic/necroticcells are shown.

FIG. 6D is a set of images of cells showing cytochrome c release inducedby BH3Is. HeLa cells were treated with 100 μM BH3I-1 for 48 hr and werethen stained with Hoechst (a) or cytochrome c antibody (b). The arrowsindicate the position of an apoptotic cell.

FIG. 6E is an image of a Western blot analysis of cytochrome c releasefrom Jurkat cells treated with BH3I-1 (100 μM) or BH3I-2 (50 μM) for 48hr. Cells were fractionated and soluble (S) and heavy membrane (M)fractions were subjected to Western blot analysis using cytochrome c(Pharmingen) and VDAC (Calbiochem) antibodies.

FIG. 6F is a graph showing the time course of caspase activation inJurkat cells, as assayed using a QuantiPak kit (Biomol). The cells weretreated with 100 μM of BH3I-1 (triangles) or 50 μM of BH3I-2 (squares)for 2, 6, 12, or 24 hr. A fluorescence based assay of caspase-3(Ac-DEVD-AMC) activity was then performed. The numbers representrelative AMC fluorescence in each sample compared to the value obtainedwith untreated Jurkat cells incubated for 24 hours, set at 1. Standarddeviation values are also shown.

FIG. 6G is a graph showing the time course of caspase activation inJurkat cells, as assayed using a QuantiPak kit (Biomol). The cells weretreated with 100 μM of BH3I-1 (triangles) or 50 μM of BH3I-2 (squares)for 2, 6, 12 or 24 hr. A fluorescence based assay of caspase-9(Ac-LEHD-AMC) activity was then performed. The numbers representrelative AMC fluorescence in each sample compared to the value obtainedwith untreated Jurkat cells incubated for 24 hours, set at 1. Standarddeviation values are also shown.

FIG. 6H is a graph showing the comparison of caspase-9 (Ac-LEHD-AMC)(open bars) and caspase-8 (Ac-IETD-AMC) (closed bars) activation 12 and24 hr after treatment of Jurkat cells with BH3Is.

FIG. 7A is a graph showing the effects of BH3Is on cell death, measuredby an MTS assay and propidium iodide staining.

FIG. 7B is a graph showing the effect of the relative cytotoxicities ofBH3I-1s, determined by propidium iodide staining, and their bindingaffinities.

FIG. 7C is a graph showing the effect of the relative cytotoxicities ofBH3I-2s, determined by propidium iodide staining, and their bindingaffinities.

FIG. 8A is a graph showing the FRET ratio of Bax/Bcl-xL binding. Thevalues represent FRET ratios normalized relative to the FRET valueobtained for the separate expression of Bax-YFP and Bcl-xL-CFP, whichwas set at 1.0. The index of cell viability, normalized relative to theDMSO treated Bcl-xL-CFP and Bax-YFP co-transfected cells, which was setas 100% viability, is shown.

FIG. 8B is a graph showing a time course of changes in FRET values andcell death, measured by propidium iodide staining, induced by BH3I-2 at50 μM.

FIG. 8C is an images of a the expression of Bad and Bcl-xL in 293 cellsco-transfected with HA-Bad and Bcl-xL-T7. After 48 hr, cells wereharvested and isolated mitochondria were treated with BH3Is for 2 hrs.Following treatment, mitochondria were pelleted by centrifugation andsubjected to Western blot analysis using anti-HA and anti-T 7antibodies.

FIG. 8D is a series of images of cells showing the effects of BH3Is onthe localization of Bad.

FIG. 9A is a graph showing the attenuation of BH3I cytotoxicity byBcl-xL, as determined by an MTS assay, in Jurkat cells or in Jurkatcells overexpressing Bcl-xL.

FIG. 9B is a graph showing the attenuation of BH3I cytotoxicity byBcl-xL, as determined by propidium iodide staining, in Jurkat cells orin Jurkat cell overexpressing Bcl-xL.

FIG. 9C is a series of graphs showing DNA fragmentation analysis ofJurkat cells overexpressing Bcl-xL and treated with BH3Is.

FIG. 9D is a series of images showing caspase activation in Jurkat cellsor in Jurkat cells overexpressing Bcl-xL and treated with BH3Is.

FIG. 9E is a graph showing the attenuation of cell death in FL 5.12cells by Bcl-xL overexpression in cells treated with BH3Is.

FIG. 9F is a graph showing the attenuation of cell death in FL 5.12cells by Bcl-xL overexpression in cells subjected to IL-3 deprivation.

FIG. 9G is a graph showing the inactivation of the anti-apoptoticactivity of Bcl-xL by BH3Is in FL 5.12 cells, as determined by propidiumiodide staining.

FIG. 9H is a graph showing the inactivation of the antiapoptoticactivity of Bcl-xL by BH3Is in FL 5.12 cells, as determined by countingcells with activated caspases.

FIG. 10A is a graph showing the comparison of BH3Is and Bax induced celldeath. HeLa cells were transfected with YFP-Bax and pEYFP-C 1 for 48 hrin the absence (open bars) or presence (closed bars) of 100 μM zVAD-fmk.YFP transfected cells were also treated with BH3I-1 (100 μM) or BH3I-2(50 μM) for 48 hours. Cell viability was determined by propidium iodide(PI) staining and microscopic examination of PI/YFP positive cells.Numbers represent the percentage of PI positive cells in the YFPpositive population. The values of standard deviation are also shown.

FIG. 10B is a graph showing the attenuation of BH3Is' cytotoxicities bythe pan-caspase inhibitor zVAD-fmk. Jurkat cells were treated withBH3I-1 or BH3I-2 in the presence (open bars) or absence (closed bars) of100 μM zVAD-fmk (Alexis) for 48 hr. Cell death was determined using anMTS assay. Numbers represent the percentages of cell death, normalizedrelative to a DMSO treated control, which was set at 100% viability.Standard deviation values are also shown.

FIG. 10C is a set of images of cells showing that nuclear fragmentation,but not cytochrome c release, induced by BH3Is, requires caspases. HeLacells were treated with 100 μM BH3I-1 and 100 μM zVAD-fmk for 48 hr andstained with Hoechst (a) or cytochrome c antibody (b). Arrows indicatethe positions of the dying cells.

FIG. 10D-1-10D-6 are a series of graphs showing DNA fragmentationanalysis of Jurkat cells treated with BH3Is and zVAD-fmk. Cells weretreated with DMSO (blank; 1 and 2), BH3I-1 (100 μM; 3 and 4), or BH3I-2(30 μM; 5 and 6) in the presence (1, 3, and 5) or absence (2, 4, and 6)of zVAD-fmk for 72 hr. The cells were then fixed and stained withpropidium iodide (PI). Samples were analyzed by FACS, and the percentageof subG1 DNA is shown for each sample.

FIG. 11 is a schematic representation of the chemical structure of acompound used to increase cell death. Each of R₁, R₂, R₄, and R₅ isindependently selected from the group consisting of hydrogen, alkoxyl,hydroxyl, and halogens; R₃ is selected from the group consisting ofN(CH₃)₂, phenyl, hydroxyl, alkoxyl, and halogens; R₆ is selected fromthe group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₃;R₇ is either hydrogen or an alkyl group; and the bond (a) is either asingle or double bond.

FIG. 12 is a schematic representation of the chemical structure of acompound used to increase cell death. Each of R₁, R₂, R₄, and R₅ is,independently, hydrogen, a halogen, or a phenyl group; and R₃ ishydrogen or an alkyl group.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods for increasing cell death, as well as fortreating a condition in a subject. Techniques for carrying out themethods of the invention are now described in detail.

Identification of Chemical Compounds that Inhibit Heterodimerization ofBcl-2 Protein Family Members

It has been well established that members of the Bcl-2 family areinvolved in the regulation of cell viability and cell death. The precisemechanism through which this regulation occurs has not been fullyelucidated. However, it is known that various Bcl-2 family members candimerize with each other. Bcl-2 family members that promote cellviability can form homodimers or heterodimers with otherviability-promoting family members, or can form heterodimers withapoptosis-promoting family members. It has been proposed that theability of a cell to undergo apoptosis depends on the level ofpro-apoptotic proteins within a cell which are not heterodimerized to aviability promoting protein such as Bcl-2 or Bcl-xL (Taylor et al., Nat.Biotech. 17:1097-1100, 1999; Kang et al., Neurosci. Lett. 256:8928-35,1998; Shibata et al., EMBO J. 18:2692-2701, 1999; Thomas et al. Oncogene12:1055-1062, 1996; Miyashita et al., Oncogene 9:1799-1805, 1994; andMotoyama et al., Science 267:1506-1510, 1995). The greater the amount ofundimerized pro-apoptotic protein in a cell, the greater the likelihoodis that the cell will die.

To tip the scales of this pathway in favor of cell death, one mayincrease the amount of free pro-apoptotic protein in a cell. This may bedone by preventing the heterodimerization of an apoptosis-promotingprotein and an anti-apoptosis-promoting protein, for example, byadministering a small molecule inhibitor of such an interaction to acell. Furthermore, it has been suggested through NMR studies, thatheterodimerization between anti-apoptotic proteins and pro-apoptoticproteins is mediated through the Bcl-2-homology-3 domain, or BH3 domain,of the pro-apoptotic Bcl-2 family members and the Bcl-2-homology-1, 2,and 3 domains (BH1, BH2, and BH3 domains) of the anti-apoptotic Bcl-2family members. For example, the pro-apoptotic protein Bak interactswith the anti-apoptotic protein Bcl-xL through its BH3 domain (Sattleret al. Science 275:983-986, 1997). Therefore, a small molecule thatinhibits the interaction of anti-apoptotic proteins and pro-apoptoticproteins would be useful for promoting cell death.

We have used a fluorescence polarization assay to screen a library of16,000 chemical compounds (ChemBridge, San Diego, Calif.) to identifysmall molecules that inhibit the interaction between a peptidecorresponding to the BH3 domain of a pro-apoptotic protein and arecombinant Bcl-xL/GST fusion protein. In this assay, an eighteen aminoacid peptide corresponding to the BH3 domain of Bak was used. The Bakpeptide was labeled with a fluorescent tag, such as OREGON GREEN. Uponbinding of the tagged peptide to the Bcl-xL/GST fusion protein, thepolarization of light emitted by the tagged peptide is altered.Therefore, in a sample in which a compound from the chemical librarydoes not inhibit the interaction between the Bak peptide and theBcl-xL/GST fusion protein the polarization of light emitted by thepeptide will be altered. Conversely, in a sample in which a compounddoes inhibit the interaction between the Bak peptide and the Bcl-xL/GSTfusion protein the polarization of light emitted by the peptide will notbe altered.

The interaction between two polypeptides may be assessed by other meansknown to those skilled in the field of molecular biology. For example,the interacting proteins may be co-immunoprecipitated using an antibodythat recognizes either of the polypeptides, using methods commonly knownin the art.

Similar assays can be performed using other members of the Bcl-2 family.For example, other pro-apoptotic Bcl-2 family members that contain a BH3domain may be substituted for the Bak polypeptide in the above-describedassay. Alternatively, peptides from other anti-apoptotic Bcl-2 familymembers may be substituted for Bcl-xL in the fusion protein in the aboveassay. Carrying out this assay using various Bcl-2 family members willidentify compounds that can be used to increase cell death.

Identification of Compounds that Inhibit Heterodimerization of Bcl-2Family Proteins and Increase Cell Death

Compounds from the chemical library identified to inhibit theinteraction between a Bak peptide and a Bcl-xL/GST fusion protein, orother pro-apoptotic/anti-apoptotic Bcl-2 family member interactions, asdescribed above, may also increase cell death. To determine this, a cellmay be administered a compound and the level of cell death that occursmay be measured and compared to the level of cell death that occurs in acell which was not administered the compound. The level of cell deathmay be measured, for example, by determining cellular ATP levelsaccording to the methods of Crouch et al. (supra), Storer et al.(supra), or Cree et al. (supra), wherein a decrease in the cellular ATPlevel indicates an increase in cell death. Alternatively, cell death maybe measured by staining a cell with a vital dye, such as trypan blue,wherein the staining of a cell with a vital dye indicates that the cellis dead. Accordingly, if a population of cells receiving a candidatecompound exhibits increased cell death, relative to an untreated controlpopulation of cells, then the candidate compound increases cell death.

Various types of cells may be used to carry out the invention. Forexample, immortalized cells, such as HeLa cells, U-937 cells, and Jurkatcells may be used. Alternatively, cells may be transfected with atransgene that promotes resistance to cell death, and used in theinvention. For example, HeLa cells may be stably transfected with aviability-promoting member of the Bcl-2 family, for example Bcl-xL.

Structural Derivatives of Chemical Compounds that Increase Cell Death

The small molecules identified to increase cell death may bestructurally modified and subsequently used to increase cell death, orto treat a subject with a condition in which the occurrence of celldeath is too low. For example, the small molecules may be modified byany of the following processes: substitution of a valine moiety withleucine, isoleucine, or alanine; substitution of a heterocyclic ringwith a benzyl ring; reduction of a double bond attached to aheterocyclic ring; introduction of additional constituents, for example,hydroxyl, alkoxyl, or halogen groups at various positions of a benzylring; substitution of bromine or chlorine moieties with a hydroxyl,alkoxyl, or phenyl group or derivative of a phenyl group; conversion ofa carboxyl group into an ester; elimination of various halogen groups;etherification of a hydroxyl group; or substitution of a halide groupwith a phenyl group, or a derivative of a phenyl group.

Therapy

A compound identified as capable of increasing cell death by any of theabove-described methods may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer theidentified compound to patients suffering from a disease in which thereis a lack of cell death. Administration may begin before the patient issymptomatic. Any appropriate route of administration may be employed,for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration. Therapeutic formulations may be in the form ofliquid solutions or suspensions; for oral administration, formulationsmay be in the form of tablets or capsules; and for intranasalformulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (19th ed.,ed. A. R. Gennaro AR., 1995, Mack Publishing Company, Easton, Pa.).Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for compounds thatincrease cell death include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

If desired, treatment with a compound identified according to themethods described above, may be combined with more traditional therapiesfor the disease characterized by a lack of cell death, for example,traditional chemotherapeutic agents, radiation therapy, or surgery.

The methods of the instant invention may be used to increase cell deathas described herein in any mammal, for example, humans, domestic pets,or livestock.

The following examples are provided to illustrate the invention. Theseexamples should not be construed as limiting.

EXAMPLE 1 Identification of Small Molecules that Inhibit InteractionBetween a Bak Polypeptide and a Bcl-xL/GST Fusion Protein

A nineteen amino acid Bak peptide, corresponding to the BH3 domain ofBak, with the sequence KGGGQVGRQLAIIGDDINR (SEQ ID NO: 2) was labeledwith the fluorophore OREGON GREEN according to the manufacturer'sinstructions (Molecular Probes, Eugene, Oreg.). In addition, arecombinant Bcl-xL/GST fusion protein was generated using conventionaltechniques known to those skilled in the fields of biochemistry andmolecular biology. The interaction between the Bak peptide and theBcl-xL/GST fusion protein was confirmed.

A screen of approximately 16,000 compounds from the ChemBridge chemicalcompound library was conducted to identify compounds that disrupt theinteraction between the Bak peptide and the Bcl-xL/GST fusion protein.The ability of a compound to disrupt the interaction between the twopolypeptides was assessed using a fluorescence polarization assay. TheBak peptide and Bcl-xL/GST fusion protein (15 nM each; dissolved in PBS)were first combined, and then the compound was added. Alternatively, theBak peptide, Bcl-xL/GST fusion protein, and test compound (5 mg/ml;dissolved in 0.1-0.5 gl of DMSO) may be combined in any order. Normallythe unbound Bak polypeptide results in polarization of approximately 40mP units. Upon binding of the Bcl-xL/GST fusion protein, polarizationincreased to approximately 100-120 mP units.

In a preferred method, the fluorescence assay was carried out asfollows. Bak BH3 peptide (Research Genetics) was labeled usingNHS-OREGON GREEN (Molecular Probes) and purified by HPLC. For theinitial screening assays, 33 nM of labeled BH3 peptide, 2 μM ofGST-Bcl-xL protein, 0.1% bovine gamma globulin (BGG, Sigma) and 1 mM DTTmixed with PBS, pH 7.2 (Gibco) were added into 384 well black plates(Lab Systems) using MULTIDROP (Lab Systems). Small molecules (5 mg/ml inDMSO, ChemBridge) were transferred using plastic 384-pin arrays(Genetix). The plates were incubated for 1-2 hr at 25° C., and thefluorescence polarization assay values were determined using an Analystplate reader (LJL Biosystems). Reactions containing 16.65 nM of labeledBH3 peptide and 4.2 μM of Bcl-xL-His₆ fusion protein, which waspreviously utilized to characterize BH3/Bcl-xL binding (Sattler et al.,Science 275:983-6, 1997) were used for the further fluorescencepolarization analyses. Kd and Ki determination was performed aspreviously described using GRAPHPAD PRISM software package (GraphPad)(Dandliker et al., Methods Enzymol. 74:3-28,1981).

In a primary screen of the approximately 16,000 chemical compounds fromthe ChemBridge library, approximately 10 compounds were identified todisrupt the interaction between Bak and the Bcl-xL/GST fusion protein.Each of these compounds had a Ki value of 50 μM or lower. Thesecompounds were selected for a secondary screen. Chemicals for furthertesting were obtained from ChemBridge, except for BH3I-1′″, which wasobtained from Chemical Diversity.

In a further screen, various concentrations of each of theabove-identified compounds were evaluated for their potency. A serialdilution of each chemical compound was performed and the fluorescencepolarization assay was repeated, as per the primary screen. As a resultof the secondary screening, three compounds, chemical compound IDnumbers 275805 (BH3I-1; FIG. 1), 282986 (BH3I-1′; FIG. 2), and 175362(BH3I-2; FIG. 3), collectively termed “BH3Is,” were identified todisrupt the interaction between Bak and the BCL-x_(L)/GST fusionprotein. Each of these compounds displayed a Ki value of 10-20 μM in thefluorescence polarization assay. Additional homologues of the BH3I-1s(BH3I-1″ and BH3I-1′″) and BH3I-2 (BH3-2″ and BH3I-2″) were alsoanalyzed in the study (FIG. 4). According to the results of thefluorescence polarization assays, the affinity of the inhibitors was inthe low μM range, with affinities decreasing in the following order:BH3I-1>BH3I-1′>BH3I-2′>BH3I-2>BH3I-2″>BH3I1″. The affinity of BH3I-1′″using the fluorescence polarization assay was not assessed, because ofits intrinsic fluorescence.

Example 2 BH3Is Inhibit Bcl-xL Heterodimerization in Vitro

To test the possibility that BH3Is target the OREGON GREEN moiety of thefluorescently labeled BH3 peptide, a novel BH3/Bcl-xL binding assayusing unlabeled BH3 peptide was designed. In this assay Bcl-xL wascovalently attached to a surface-enhanced laser desorption/ionization(SELDI) chip, and binding of the unlabelled BH3 peptide to theimmobilized protein was monitored by mass spectrometry. Specifically,purified recombinant GST-Bcl-2 and BCL-xL-His₆ were coupled throughtheir primary amines to SELDI chip surfaces derivatized withcarbonyldiimidazole (Ciphergen). Bak BH3 peptide was then added (in atotal volume of 1 μl) for 12 hours at 4° C. in a humidified chamber, toallow binding to each spot of the SELDI chip. The chip was then washedwith alternating high and low pH-0.1M sodium acetate with 0.5M NaCl,followed by 0.01M HEPES; pH 7.3. The samples were embedded inα-cyanno-4-hydroxycinnamaic acid matrix and analyzed for mass by MALDITOF. An average of 100 laser shots at a constant setting were collectedover 20 spots in each sample.

BH3 peptide bound to the Bcl-xL-modified surface in a dose-dependentfashion (FIG. 5A). Addition of BH3I-1 or BH3I-2 resulted in thereduction of BH3 binding. BH3I-2 had higher activity than BH3I-1 in thisassay. Similar to the fluorescence polarization assay results, BH3I-1″showed lower potency than BH3I-1 in this assay. In addition, BH3Isdisrupted the BH3/Bcl-2 interaction (FIG. 5B). In this study, Bak BH3peptide was incubated with SELDI surface modified with 5 pmoles ofGST-Bcl-2 in the presence or absence of BH3Is. As a negative control BakBH3 peptide was incubated with SELDI surface without Bcl-xL/Bcl-2proteins. These data suggest that BH3Is can target multipleanti-apoptotic Bcl-2 family members.

Next, it was determined whether BH3Is could disrupt theheterodimerization of Bcl-xL with pro-apoptotic proteins from the Bcl-2family, such as truncated Bid (tBid). Truncation of Bid by caspase-8activates Bid and dramatically increases its affinity towardsanti-apoptotic Bcl-2 family members (Li et al., Cell 94:491-501, 1998).To perform this study, in vitro translation of tBid or U2AF⁶⁵ (control)was performed using TNT® Coupled Reticulocyte and Wheat Germ LysateSystems (Promega) respectively. Bcl-xL, U2AF³⁵ (control) orcorresponding amounts of original Ni-NTA agarose beads (blank) werepre-incubated with the BH3 inhibitors or peptide in 100 μl of PBS for 30min at 25° C. Then 1 μl of ³⁵S-labeled tBID or U2AF⁶⁵ was added andincubation was continued for 2 hr at 4° C. The samples were subjected toSDS-PAGE, coomassie blue staining and autoradiography.

In vitro translated tBid specifically bound to His-tagged Bcl-xLimmobilized on Ni²⁺ beads (FIG. 5C). Addition of either BH3I-2 or BH3I-1resulted in a dose-dependent decrease in the tBid binding. BH3I-1 showedlower activity than BH3I-2 (FIG. 5D), and the activity of BH3I-1″ waseven lower.

The results of the tBid pulldown and SELDI assays indicate that the useof OREGON GREEN moiety in fluorescence polarization assays could haveresulted in an overestimation of the Ki values of the BH3I-1s relativeto BH3I-2s. To verify this conclusion, NMR titrations of Bcl-xL withBH3I-1s were performed and their Ki values were determined (FIG. 4). Theresults of NMR titrations showed that the relative order of affinitiesof BH3I-1s is: BH3I-1>BH3I-1′>BH3I-1″>BH3I-1′″. Moreover, the NMRderived Ki value of BH3I-1 was lower than the fluorescence polarizationassay derived Ki value of BH3I-2. The Ki value for BH3I-2 was not ableto be verified using NMR titrations because of the predominantlyintermediate exchange rate displayed by this compound. The BH3I-1s Kivalues obtained by the NMR approach agree with the results of bothpulldown and SELDI analyses. Therefore, the order of affinities of BH3Isis: BH312′>BH3I-2>BH3I-2″>BH3I-1>BH3I-1′>BH3I-1″>BH3I-1′″.

EXAMPLE 3 BH3Is Are Selective Inhibitors of Bcl-2 Family Proteins

To determine the specificity of BH3Is, their ability to inhibit otherprotein-protein interactions was tested using the pulldown assaydescribed above and the splicing factors U2AF³⁵ and U2AF⁶⁵ (Zhang et al,Proc. Natl. Acad. Sci. USA 89:8769-73, 1992). Neither BH3I-1 nor BH3I-2affected the interaction between the splicing factors (FIG. 5E). BH3Is(80 μM) also had no effect on the interaction between the Apaf-1 CARDdomain fragment and caspase-9 in the SELDI assay.

Since NMR spectroscopy allows the detection of low-affinity (up to Kd=1mM) interactions, it was used to confirm the absence of inhibition ofthe control protein-protein interactions by BH3Is. BH3Is did not affectthe interactions between the CIDE-N domains of CIDE-B and DFF40 or DFF45(Lugovskoy et al., Cell 99:747-55, 1999). Despite close structuralhomology between full length Bid and Bcl-xL, the BH3Is failed to bindBid. Therefore, these compounds demonstrate a high degree of specificity(Chou et al., Cell 96:615-24, 1999; and McDonnell et al., Cell96:625-34, 1999).

EXAMPLE 4 Small Molecules That Inhibit Interaction Between a BakPolypeptide and a Bcl-x_(L)/GST Fusion Protein Also Increase Cell Death

The BH3I compounds that were found to disrupt the interaction between aBak peptide and a BCL-xL/GST fusion protein were evaluated for theircytotoxic effects. HeLa cells, U-937 cells, or Jurkat cells, each ofwhich endogenously express both Bak and Bcl-xL, were contacted with eachof the BH3I chemical compounds identified above, by adding the compoundto the cell culture media. Forty-eight hours after the cells receivedthe chemical compound the cell culture sample was evaluated for thelevel of cell death that had occurred in response to the chemicalcompound. The level of cell death was determined by measuring cellularATP levels, wherein a decrease in cellular ATP levels indicated anincrease in cell death. In each cell line, cell death was increased inthe sample that received one of the above-identified compounds. In eachcell line, chemical compounds 275805 (BH3I-1) and 282986(BH3I-1′)displayed GI50 values of 50-70 μM, while chemical compound 175362(BH3I-2) displayed a GI50 value of 10-15 μM. In addition, the cellstreated with the above chemical compounds and subsequently stained withHoescht exhibited extensive nuclear fragmentation, indicating thatapoptosis was occurring in the cells.

To further examine the ability of BH3Is to induce apoptosis, Jurkatcells were treated with BH3I-1 (100 μM) (FIG. 6A, panels a and b),BH3I-2 (30 μM) (FIG. 6A, panels c and d) or DMSO (FIG. 6A, panels e andf) for 48 hr. The cells were then stained using Hoechst dye and a FragELTUNEL kit (Oncogene Research Products). The treatment resulted in TUNELpositivity, indicating that the BH3Is induced DNA fragmentation. Inaddition, a significant portion of the cells treated with a BH3Idisplayed sub-G1 DNA content, which is indicative of apoptosis (FIG.6B). This was determined by treating Jurkat cells with BH3I-1 (100 μM)or BH3I-2 (30 μM), fixing them 72 hr later, and staining them withpropidium iodide (PI). The samples were analyzed by FACS. Finally, BH3Isinduced the appearance of Annexin V binding without an increase in PIstaining (FIG. 6C). These results demonstrate that treatment of cellswith a BH3I induces apoptosis.

Next, the role of BH3 Is in inducing the release of cytochrome c frommitochondria was examined. Immunostaining with cytochrome c antibody andfractionation experiments showed that BH3I-1 (FIGS. 6D and 6E) andBH3I-2 (FIG. 6E) induced cytochrome c release. DiOC₆ staining failed todemonstrate a decrease in mitochondrial membrane potential in cellstreated with BH3Is for 48 hr.

To evaluate the activation of caspases by BH3Is, the activation ofcaspases in the presence of these compounds was measured using thepreferred caspase-3 and caspase-9 fluorogenic substrates DEVD.amc andLEHD.amc. Treatment of Jurkat cells with either BH3I-1 or BH3I-2resulted in an increase in caspase-3-like (FIG. 6F) and caspase-9-like(FIG. 6G) activities. Activation of both caspases followed anessentially identical time course and reached a maximum 12 hours afteraddition of the BH3I. If BH3Is induce apoptosis through a mitochondrialpathway, caspase-9 should be activated before caspase-8, since thelatter is a mediator of the death receptor pathway at the plasmamembrane (Cryns et al., Genes Dev. 12:1551-70, 1998 [published erratumappears in Genes Dev. 13:371, 1999]). Therefore, the levels of caspase-9and caspase-8 activity were compared by measuring LEHD.amc/IETD.amccleavage activities. The BH3Is failed to induce the activation ofcaspase-8-like activity after 12 and 24 hrs of treatment whencaspase-9-like activity was elevated (FIG. 6H). These data indicate thatBH3Is induce the activation of caspases in the mitochondrial pathway.

The conditions under which cell death occurs in the presence of BH3Iswere further evaluated. HeLa cells were pretreated with zVAD-fmk (100μM) for 1 hour. The purpose of such a treatment was to inhibit theintracellular pathways mediating cell death by apoptosis. Each of thechemical compounds BH3I-1, BH3I-1′, or BH3I-2 was then added to themedia of the cells of three separate samples. After 48 or 72 hours ofexposure of the cells to zVAD-fmk and the chemical compound, the levelof cell death was measured for each sample, by measuring cellular ATPlevels. In each cell sample, the cellular ATP levels decreased relativeto samples that received no chemical compounds from the ChemBridgelibrary. These results indicated that the above-identified compoundsexert their effects on cell death not only through apoptosis, but alsothrough necrosis.

EXAMPLE 5 Small Molecules That Inhibit Interaction Between a BakPolypeptide and a Bcl-xL/GST Fusion Protein Also Increase Cell Death inCells Resistant to Cell Death

In another study, HeLa cells that stably overexpress human BCL-XL, andare therefore more resistant to cell death, were pretreated withzVAD-fmk (100 μM) for 1 hour, as described above. Each of the chemicalcompounds BH3I-1, BH3I-1′, and BH3I-2 from the ChemBridge library wasthen added to the media of the cells of three separate samples. After 48or 72 hours of exposure of the cells to zVAD-fmk and the chemicalcompound, the level of cell death was measured for each sample, bymeasuring cellular ATP levels. In each cell sample, the cellular ATPlevels decreased relative to samples that received no chemical compoundsfrom the ChemBridge library. These results demonstrate that cells whichare resistant to cell death undergo death in response to BH3I-1,BH3I-1′, and BH3I-2.

EXAMPLE 6 BH3Is Induce Apoptosis Through Disruption of the BH3 DomainInteraction

Since BH3Is induce apoptosis, their mode of action, for example, throughdisruption of BH3 domain interactions, was investigated next. Ifapoptosis induced by a BH3I is BH3-dependent, then cytotoxicity shouldcorrelate with the compound's ability to disrupt BH3 domain interactionsin vitro. To examine this possibility, Jurkat cells were treated withthe BH3 Is and the occurrence of cell death was assayed by the MTS assayand propidium iodide (PI) uptake (FIG. 7A). The cytotoxicity of BH3-Isfollowed the order ofBH3I-2′>BH3I-2>BH3I-2″>BH3I-1>BH3I-1′>BH3I-1″>BH3I-1′″ which paralleledthe order of their Ki values determined using in vitro Bcl-xL bindingassays (FIGS. 7B and 7C). Similar data were obtained using a trypan blueexclusion assay. These results suggest that the ability to inhibit theBH3 domain interaction is critical for BH3I-mediated induction of celldeath.

The effect of the BH3Is on the disruption of the heterodimerization ofBcl-2 family proteins in vivo was next determined. Dimerization of Bcl-2in immunoprecipitation experiments may represent post-lysis eventsinduced by detergents (Hsu et al., J. Biol. Chem. 272:13829-34, 1997).Therefore, these studies focused on assaying the Bcl-xL dimerizationstatus in intact cells. Recently, the interaction between Bax and Bcl-2has been studied in intact living cells by fluorescent resonance energytransfer measurements between GFP fusion proteins (Mahajan et al., Nat.Biotechnol. 16:547-52, 1998). This approach was used to monitor theeffects of BH3Is on Bcl-xL/Bax heterodimerization in intact cells.

293 cells were transfected with Bcl-xL-CFP and Bax-YFP expressionvectors using Lipofectamine Plus (Gibco) or TransLT-1 (PanVera) and 24hr later were treated with BH3Is. Cells were harvested in PBSsupplemented with BH3Is, and fluorescence was determined using a C-60fluorimeter (PTI) or a Wallac platereader. Fluorescence in the samplesseparately overexpressing Bax and Bcl-xL was added together and used toestimate the FRET value in the absence of dimerization. The extent ofFRET between CFP and YFP was determined as a ratio of fluorescence at527 nm vs. 475 nm after excitation at 433 nm.

Co-transfection of Bax-YFP and Bcl-xL-CFP resulted in an increase in therelative FRET ratio from 1.0 (when two proteins are expressedseparately) to 1.7 (FIG. 8A), indicative of Bax/Bcl-xL interactions inthe cells. Addition of BH3Is resulted in changes in the FRET ratioconsistent with the in vitro activities of the compounds:BH3I-2>BH3I-1>BH3I-1″. YFP 513/527 nm fluorescence was not affected byFRET and could serve as an accurate indicator of the Bax-YFP levels anda rough index of cell death. The relative effects on YFP fluorescencereduction also followed the order of BH3I-2>BH3I-I>BH3I-1″ (FIG. 8A).These results are consistent with previous cell viability studies anddemonstrate that the ability of the BH3Is to disrupt Bax/Bcl-xLinteractions in intact cells directly correlates with cytotoxicity inthe same samples. Time course experiments, which demonstrated thatdisruption of FRET preceded cell death, were also performed (FIG. 8B).

Bad is a pro-apoptotic member of the Bcl-2 family, whoseapoptosis-promoting activity and mitochondrial targeting depends onheterodimerization with anti-apoptotic Bcl-2 family members (Zha et al.,Cell 87, 619-28, 1996). To examine the effects of BH3Is on Badlocalization, BSC-1 cells were transfected with 5 μg of Bad-GFPexpression vector alone (FIG. 8D, panels 1 and 5) or were co-transfectedwith 5 μg of Bad-GFP and 5 μg of Bcl-xL-T7 (FIG. 8D, panels 2-5). Twentyfour hours after transfection, cells were treated with BH3I-1 (100 μM;FIG. 8D, panel 4) or BH3I-2 (50 μM; FIG. 8D, panel 5) for 6 hr. The GFPsignal in the fixed cells is shown in FIG. 8D, panels 1 and 3-5. Inorder to determine mitochondrial localization, cells were pre-stainedwith MitoTracker Red CMXRos for 30 min prior to fixation (FIG. 8D, panel2). All cells in this study were pretreated with 100 μM zVAD to decreasethe detrimental effects of overexpression of proapoptotic Bcl-2 proteinson cell viability.

Transfection of the cells with the Bad expression vector resulted in thepredominantly cytoplasmic localization of Bad (FIG. 8D, panel 1),whereas co-transfection with Bcl-xL resulted in the mitochondriallocalization of Bad (FIG. 8D, panel 2 and 3). BH3Is disrupted themitochondrial association of Bad, but not Bcl-xL, after in vitrotreatment of the mitochondria isolated from 293 cells co-transfectedwith Bad and Bcl-xL (FIG. 8C). The relative activities of the BH3Is inthis assay (BH3I-2>BH3I-1>BH3I-1″) were consistent with the previousresults. Treatment of cells with either BH3I-1 or BH3I-2 resulted in anincrease in the number of cells with the cytosolic redistributedBad-GFP. Quantitation of cells with mitochondrial versus cytosoliclocalization of Bad-GFP indicated that BH3I-1 and BH3I-2 efficientlydisrupted mitochondrial targeting and heterodimerization of Bad-GFP witheither Bcl-2 or Bcl-xL in intact cells (FIG. 8D). Again, the order ofthe activities in these assays was BH3I-2>BH3I-1>BH3I-1“. No cell deathwas detectable at the time of measurement, suggesting that in thissystem, disruption of BH3 interactions also precedes cell death.

If BH3Is act through inhibiting the function of anti-apoptotic membersof Bcl-2 family, overexpression of Bcl-xL should provide protectionagainst BH3Is, which can be overcome by increased amount of BH3Is.Furthermore, Bcl-xL overexpressing cells. should still undergo apoptosiswhen treated with a high enough dose of a BH3I to overcome theprotection by Bcl-xL. To test this theory, Jurkat/Bcl-xL cells weretreated with BH3I-1 and BH3I-2 for 48 hr. Cell death was determinedusing the MTS assay (FIG. 9A) or PI staining (FIG. 9B). Overexpressionof Bcl-xL provided protection for Jurkat cells from lower doses of BH3Is(up to 40 μM of BH3I-2 and up to 100 μM of BH3I-1). Overexpression ofBcl-xL, however, gave no protection after treatment with 80 μM of BH3I-2or 200 μM of BH3I-1. BH3I treatment (100 μM of BH3I-1 or 30 μM ofBH3I-2) induced the appearance of sub-GI DNA, which is also indicativeof apoptosis (FIG. 9C). To determine if caspases were activated inJurkat/Bcl-xL cells treated with high doses of BH3Is, indicating thatcells were undergoing apoptosis rather than necrosis, the cell werestained with FAM-VAD-fik, which binds to the active caspases and allowsvisualization of caspase activation in intact cells (FIG. 9D). Caspaseswere activated in Hoechst-positive dying cells following treatment withhigh doses of BH3Is. No Hoechst/caspase positive cells were seen inuntreated control cells.

In an additional study of the attenuation of cell death by Bcl-xLoverexpression and treatment with BH3Is, FL5.12 cells undergo apoptosisin response to IL-3 deprivation, and this cell death is efficientlyblocked by overexpression of Bcl-xL (Vander Heiden et al., Mol. Cell.3:159-67, 1999) (FIG. 9F). This effect could be overcome by a higherdose of BH3Is (FIG. 9E). Inactivation of Bcl-xL by BH3Is inFL5.12/Bcl-xL cells was detected as an increase in cell death followingIL-3 deprivation. The cytotoxicity of BH3I-1 and BH3I-2 significantlyincreased upon IL-3 deprivation of the Bcl-xL overexpressing cells,whereas IL-3 deprivation alone did not induce significant cell death(FIG. 9G). Moreover, staining cells with FAM-VAD-fmk revealed increasedcaspase activation induced by combined treatment with BH3Is and IL-3deprivation in Bcl-xL/FL5.12 cells (FIG. 9H), demonstrating anenhancement in apoptosis in these cells in response to Bcl-xLinactivation.

Finally, if inhibition of the BH3 domain interaction results in therelease of pro-apoptotic members of Bcl-2 family, the pro-apoptoticactivity of BH3Is should mimic that of pro-apoptotic members of theBcl-2 family. Therefore, the pro-apoptotic activity of the BH3Is wascompared with that of Bax. Cell death induced by either BH3I treatmentof HeLa cells or by transfection with Bax was only partially dependentupon caspase activity (FIG. 10A). MTS (FIG. 10B) and PI (FIG. 10A)exclusion assays of BH3Is/zVAD treated Jurkat cells confirmed thisconclusion.

In order to further characterize the role of caspases in BH3I-inducedapoptosis, HeLa cells that had been treated with BH3Is in the presenceof zVAD-fmk were stained with Hoechst dye and anti-cytochrome cantibody. Addition of zVAD-fmk completely protected cells fromBH3I-induced nuclear fragmentation, but not from nuclear condensation orcytochrome c release induced by BH3I-1 (compare FIG. 10C and FIG. 6D)and BH3I-2. This conclusion was confirmed by subG1 analysis showing thatzVAD-fmk prevented the appearance of fragmented DNA in cells treatedwith BH3Is (FIGS. 10D1-10D6). Therefore, although BH3Is induce someevents that require caspase activation, similar to Bax-induced celldeath, eventual cellular demise is only partially dependent upon caspaseactivity (Xiang et al., Proc. Natl. Acad. Sci. USA 93:14559-63,1996; andGross et al., EMBO J. 17:3878-85, 1998). Thus, BH3Is induce apoptosis bydisrupting the BH3 domain interaction between pro- and anti-apoptoticmembers of the Bcl-2 family.

EXAMPLE 7 Neither Bak BH3 Peptide nor BH3Is Affect the Pore Formation byBcl-xL

Bcl-xL forms membrane pores, and the formation of these pores may play arole in Bcl-xL's ability to regulate apoptosis. Thus, the effects of theBH3Is on pore formation by Bcl-xL were tested (Minn, et al., Nature385:353-7, 1997; Schendel et al., Proc. Natl. Acad. Sci. USA 94:5113-8,1997; Minn et al., EMBO J. 18:632-43,1999; and Matsuyama et al., J.Biol. Chem. 273:30995-1001, 1998). Liposomes containing 20 mM of5,6-carboxyfluorescein were prepared as previously described (Antonsson,et al., Science 277:370-2,1997). For Bcl-xL pore formation 5 μMBcl-xL-His₆ was preincubated with BH3I-2 or Bak BH3 peptide in 50 μl of5 mM sodium citrate, 150 mM NaCl, pH 4.0 buffer for 10 min at 25° C.,followed by addition of 5 μl of undiluted liposomes. Prior todetermination of the fluorescence pH was adjusted by adding 10 μl of 1.5M Tris-HCl, pH 7.5. Neither BH3Is nor BH3 peptide affectedBcl-xL-mediated release of carboxyfluorescein encapsulated in artificialliposomes. This result suggests that Bcl-xL pore formation isindependent of BH3-mediated homodimerization, and that the pro-apoptoticactivity of BH3Is reflects a critical role of BH3-dependentheterodimerization in mediating cell survival.

EXAMPLE 8 BH3Is Interact with the BH3-peptide Binding Pocket of Bcl-xL

NMR titration was used to examine whether BH3Iinteracts with the bindingpocket of Bcl-xL in a manner similar to Bak BH3 peptide (Hajduk et al.,Science 278:497-499, 1997).

First, the changes in 2D ¹⁵N/¹H heteronuclear single quantum correlationspectrum (HSQC) of ¹⁵N-labeled Bcl-xL upon addition of the Bak BH3peptide were analyzed. Addition of Bak BH3 peptide primarily affectedresidues in the BH1-BH3 hydrophobic cleft, especially residues on theBH1/BH2 interface, consistent with the published structure of theBak/Bcl-xL complex (Sattler et al., Science 275:983-6, 1997).

Next, changes in Bcl-xL structure upon addition of increasing amounts ofBH3Is were determined. Each of the BH3Is induced significant changes inthe Bcl-xL structure. The compounds targeted the hydrophobic cleftformed by the BH1, BH2, and BH3 domains on the surface of the Bcl-xLprotein, and primarily bound to the area formed by BH1 and BH2 domains.The NMR data also indicated that BH3I-2s display slower dissociationrates than BH3I-1s, judged by the predominantly intermediate exchangekinetics of BH3I-2s binding compared to the fast exchange shown byBH3I-1s.

Since BH3I-1 differs from BH3I-1″ by a single substitution, the changesinduced by these chemicals in the ¹⁵N/¹H HSQC spectra of Bcl-xL werecompared (Hajduk et al., J. Med. Chem. 42:2315-7, 1999). The primaryarea differentially affected by BH3I-1 and BH3I-1″ was in the middle ofthe BH2 domain (residues N100, G102, I104, A106, F110, G111, G112). Theonly other differentially-affected residue was R55, which is positionedin the BH3 domain. Similar comparative analysis of BH3I-2 and BH3I-2′resulted in the mapping of A164, A165, R168, located C-terminal to theBH1 domain, and F110 in the BH2 domain. This suggests that BH3I-2starget a more upstream part of the Bcl-xL hydrophobic groove than thatwhich is targeted by BH3I1-s.

Additionally, the magnetization transfer between the benzene ringprotons of BH3I-1 and the amide protons of Y65 and F107 in a NuclearOverhauser Effect (NOE) spectrum was observed, indicative of directcontact between BH3I-1 and these residues. Interestingly, residue F107,as well as residues F110, A164, A165, and R168 (residues identified indifferential mapping analysis) are buried in the structure of freeBcl-xL, but are surface exposed in the structure of Bcl-xL/Bak complex(Muchmore et al., Nature 381:335-41, 1996; and Sattler et al., Science275:983-6,1997). In this complex Y65, F107, and F100 form directcontacts with the side-chain of the leucine residue of Bak BH3 peptide,which is essential for binding. This observation suggests that uponbinding of BH3I-1, Bcl-xL undergoes a conformational change similar tothat induced by the Bak BH3 peptide.

Overall, the results of the above NMR studies demonstrated that BH3Istarget the hydrophobic cleft on the surface of Bcl-xL, which is adocking site for the BH3 domain of Bak that mediates the dimerization ofBcl-2 family members. Binding of BH3Is to the hydrophobic pocket ofBcl-xL affects the conformation of Bcl-xL in a fashion similar to thatof the Bak BH3 peptide binding. This suggests a similarity between themodes of action of the BH3Is and the Bak BH3 peptide.

EXAMPLE 9 Structural Derivatives of Small Molecules that Increase CellDeath

One or more of the following modifications of the small molecules thatincrease cell death may be made and evaluated for their efficacy inincreasing cell death.

The chemical compound 275806 (BH3I-1) or 282986 (BH3I-1′) may bemodified by substituting the valine moiety with a leucine, isoleucine,or alanine moiety; substituting the heterocyclic ring with a benzyl ring(wherein substituents on the central may be in para-, meta, orortho-positions); reducing the double bond (bond (a) in FIG. 11)attached to the heterocyclic ring; introducing additional constituents,for example, hydroxyl, alkoxyl, or halogen groups at various positions(for example at each of positions R₁, R₂, R₄, and R₅ in FIG. 11) of thebenzyl ring; substituting bromine or chlorine (for example, at the R₃position in FIG. 11) with a hydroxyl, alkoxyl, or phenyl group orderivative of a phenyl group; or converting the carboxyl group (forexample, the R₇ group in FIG. 11) into an ester.

The chemical compound 175362 (BH3I-2) may be modified by eliminatingvarious halogen groups (for example each of R₁, R₂, R₄, and R₅ in FIG.12); etherification of the hydroxyl group (for example, R₃ in FIG. 12);or substitution of a halide group with a phenyl group, or a derivativeof a phenyl group (for example each of R₁, R₂, R₄, and R₅ in FIG. 12).

OTHER EMBODIMENTS

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

1. A method for increasing cell death, said method comprising the steps of: (a) providing a cell predicted to be resistant to cell death, or to be at risk for resisting cell death; and (b) contacting said cell with a chemical compound having the formula:

wherein each of R₁, R₂, R₄, and R₅ is, independently, hydrogen, a halogen, or a phenyl group; and R₃ is hydrogen or an alkyl group.
 2. The method of claim 1, wherein each of R₁, R₄, and R₅ is chlorine; R₂ is bromine; and R₃ is hydrogen.
 3. The method of claim 1, wherein each of R₁, R₄, and R₅ is chlorine; R₂ is iodine; and R₃ is hydrogen.
 4. The method of claim 1, wherein each of R₁ and R₂ is iodine; each R₄ and R₅ is chlorine; and R₃ is hydrogen.
 5. The method of claim 1, wherein said cell expresses a pro-apoptotic or anti-apoptotic protein.
 6. The method of claim 1, wherein said compound is substantially pure.
 7. The method of claim 1, wherein said compound is in a pharmaceutically acceptable carrier.
 8. The method of claim 5, wherein said pro-apoptotic protein is selected from the group consisting of pro-apoptotic proteins containing a Bcl-2-homology-domain-3.
 9. The method of claim 8, wherein said pro-apoptotic protein is selected from the group consisting of Bax, Bak, Bok, Bad, Bid, Bik, Bim, and Hrk.
 10. The method of claim 5, wherein said anti-apoptotic protein is chosen from the group consisting of Bcl-2, Bcl-xL, Mcl-1, and Bcl-w.
 11. The method of claim 1, wherein said cell is mammalian.
 12. The method of claim 11, wherein said cell is human.
 13. The method of claim 11, wherein said cell is a rodent cell. 